Method for Reducing Sequencing Errors Caused by DNA Fragmentation

Abstract

Provided herein, among other things, is a method for reducing sequencing errors caused by mechanical DNA fragmentation. In some embodiments, this method may comprise: (a) obtaining a DNA sample; (b) mechanically fragmenting the DNA sample to produce a template; (c) treating the fragmented DNA with a plurality of DNA repair enzymes; and (d) obtaining a sequence of the fragmented DNA. The treatment step (c) reduces the number of fragmentation induced errors.

What is claimed is: 
     
         1 . A method for reducing sequencing errors caused by mechanical DNA fragmentation, comprising:
 (a) obtaining a DNA sample;   (b) mechanically fragmenting the DNA sample to produce a template;   (c) treating the fragmented DNA with a plurality of DNA repair enzymes; and   (d) obtaining a sequence of the fragmented DNA,   wherein step (c) reduces the number of fragmentation induced errors.   
     
     
         2 . The method of  claim 1 , wherein step (b) is done by acoustic shearing. 
     
     
         3 . The method of  claim 1 , wherein the treating is done by treating the template with enzymes that include at least one DNA glycosylase, an AP endonuclease, a DNA polymerase and a ligase. 
     
     
         4 . The method of  claim 3 , wherein the enzymes are in a mixture. 
     
     
         5 . The method of  claim 3 , wherein the at least one DNA glycosylase comprises oxoguanine glycosylase (OGG) or formamidopyrimidine-DNA glycosylase (FPG). 
     
     
         6 . The method of  claim 1 , wherein step (d) comprises amplifying the template. 
     
     
         7 . The method of  claim 1 , wherein step (d) comprises circularlizing the template. 
     
     
         8 . The method of  claim 1 , further comprising analyzing sequence reads to identify a sequence variation. 
     
     
         9 . The method of  claim 1 , wherein the DNA sample made from a clinical sample. 
     
     
         10 . A method for detecting damaged DNA in a sample, comprising:
 preparing a sequencing library from the damaged DNA;   sequencing the sequencing library to obtain a plurality of first Watson end sequence reads (R1s) and plurality of second complementary Crick sequence reads (R2s);   for a selected locus, identifying those R1s and R2s that have a sequence abnormality; and   detecting an imbalance between the number of R1s that have a sequence abnormality and the number of R2s that have a complementary sequence abnormality at the same position.   
     
     
         11 . The method of  claim 10 , wherein the damaged DNA is from a clinical sample. 
     
     
         12 . The method of  claim 11 , wherein the clinical sample is a formalin fixed paraffin embedded (FFPE) sample. 
     
     
         13 . The method of  claim 10 , wherein the damaged DNA is fragmented. 
     
     
         14 . The method of  claim 13 , wherein the damaged DNA is fragmented by acoustic shearing. 
     
     
         15 . The method of  claim 10 , wherein the sequence abnormality is a G to T transversion. 
     
     
         16 . The method of  claim 10 , further comprising eliminating sequence reads, or sequence abnormalities, from further analysis if they are associated with an imbalance.